1. Field of the Invention
The invention generally relates to cell-specific expression vectors. It particularly relates to targeted gene therapy using recombinant expression vectors and particularly adenovirus vectors. The invention specifically relates to modulatable replication-conditional expression vectors and methods for using them. Such vectors are able to selectively replicate in a target cell or tissue to provide a therapeutic benefit in a tissue from the presence of the vector per se or from one or more heterologous gene products expressed from the vector and distributed throughout the tissue, and which vectors are designed so that replication and gene expression from the vector can be modulated.
In such vectors, a gene essential for replication is placed under the control of a heterologous tissue-specific transcriptional regulatory sequence. Thus, replication is conditioned on the presence of a factor(s) that induces transcription or the absence of a factor(s) that inhibits transcription of the gene by means of the transcriptional regulatory sequence.
Preferred vectors contain a heterologous gene that produces a product that increases or inhibits viral replication. Such genes are useful for modulating viral replication and thus also for modulating expression of genes in the vector. With these vectors, therefore, the vector can be expressed in a desirable cell, target tissue can be selectively treated, and replication and expression modulated.
The invention also relates to cells and/or methods to produce multiple heterologous gene products in high quantity from essentially one promoter element.
The invention also relates to methods of using the vectors to screen a tissue for the presence or absence of transcriptional regulatory functions that permit vector replication by means of the transcriptional regulatory sequence.
2. Background Art
The introduction of exogenous genes into cells in vitro or in vivo, systemically or in situ, has been of limited use for compositions in which it would be disadvantageous for non-target cells to take up the exogenous gene. One strategy to overcome this problem is to develop administration procedures or vectors that target a specific cell-type. Using systemic administration, attempts have been made to direct exogenous genes to myocytes and muscle cells by direct injection of DNA, to direct the exogenous DNA to hepatocytes using DNA-protein complexes, and to endothelial cells using liposomes.
Using in situ administration, retroviral replication functions have been utilized to target cells that are actively replicating.
Thus far, the ability to target cells has been limited, however, by the lack of cell-type specificity and low gene transfer efficiencies. The limited ability to target an exogenous gene to diseased cells in an organism, while avoiding (eliminating) uptake of the gene by normal, untargeted cells, particularly has been an obstacle to developing effective gene-transfer-based therapies for diseases in animals and humans.
One especially difficult challenge is targeting tumor cells. Many seemingly promising strategies for these cells, moreover, are limited to one or a few cell-types.
The present invention, in one aspect, provides a way to deliver an exogenous gene efficiently, with high distribution in a tumor and in a controlled manner.
Adenoviruses are nonenveloped, regular icosohedrons. The protein coat (capsid) is composed of 252 capsomeres of which 240 are hexons and 12 are pentons. Most of the detailed structural studies of the adenovirus polypeptides have been done for adenovirus types 2 and 5. The viral DNA is 23.85xc3x97106 daltons for adenovirus 2 and varies slightly in size depending on serotype. The DNA has inverted terminal repeats and the length of these varies with the serotype.
The replicative cycle is divided into early (E) and late (L) phases. The late phase defines the onset of viral DNA replication. Adenovirus structural proteins are generally synthesized during the late phase. Following adenovirus infection, host DNA and protein synthesis is inhibited in cells infected with most serotypes. The adenovirus lytic cycle with adenovirus 2 and adenovirus 5 is very efficient and results in approximately 10,000 virions per infected cell along with the synthesis of excess viral protein and DNA that is not incorporated into the virion. Early adenovirus transcription is a complicated sequence of interrelated biochemical events, but it entails essentially the synthesis of viral RNAs prior to the onset of viral DNA replication.
The organization of the adenovirus genome is similar in all of the adenovirus groups and specific functions are generally positioned at identical locations for each serotype studied. Early cytoplasmic messenger RNAs are complementary to four defined, noncontiguous regions on the viral DNA. These regions are designated (E1-E4). The early transcripts have been classified into an array of immediate early (E1a), delayed early (E1b, E2a, E2b, E3 and E4), and intermediate (IVa2.IX) regions.
E1a is a transactivator of multiple gene products in adenovirus through activation of the E1b, E2, E3 and E4 promoters. The E1a region is involved in transcriptional transactivation of viral and cellular genes as well as transcriptional repression of other sequences. The E1a gene exerts an important control function on all of the other early adenovirus messenger RNAs. In normal tissues, in order to transcribe regions E1b, E2a, E2b, E3, or E4 efficiently, active E1a product is required.
The E1b region is required for the normal progression of viral events late in infection. The E1b product acts in the host nucleus. Mutants generated within the E1b sequences exhibit diminished late viral mRNA accumulation as well as impairment in the inhibition of host cellular transport normally observed late in adenovirus infection (Berkner, K. L., Biotechniques 6:616-629 (1988)). E1b is required for altering functions of the host cell such that processing and transport are shifted in favor of viral late gene products. These products then result in viral packaging and release of virions. E1b produces a 19 kD protein that prevents apoptosis. E1b also produces a 55 kD protein that binds to p53.
For a complete review on adenoviruses and their replication, see Horwitz, M. S., Virology 2d ed., Fields, B. N., eds., Raven Press Limited, New York (1990), Chapter 60, pp. 1679-1721.
Adenovirus provides advantages as a vector for adequate gene delivery for the following reasons. It is a double stranded DNA nonenveloped virus with tropism for the human respiratory system and gastrointestinal tract. It causes a mild flu-like disease. Adenoviral vectors enter cells by receptor mediated endocytosis. The large (36 kilobase) genome allows for the removal of genes essential for replication and nonessential regions so that foreign DNA may be inserted and expressed from the viral genome. Adenoviruses infect a wide variety of cell types in vivo and in vitro. Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenovirus vectors rarely integrate into the host chromosome; the adenovirus genome remains as an extrachromosomal element in the cellular nucleus. There is no association of human malignancy with adenovirus infection; attenuated strains have been developed and have been used in humans for live vaccines.
For a more detailed discussion of the use of adenovirus vectors for gene therapy, see Berkner, K. L., Biotechniques 6:616-629 (1988); Trapnell, B. C., Advanced Drug Delivery Reviews 12:185-199 (1993).
Adenovirus vectors are generally deleted in the E1 region of the virus. The E1 region may then be substituted with the DNA sequences of interest. It was pointed out in a recent article on human gene therapy, however, that xe2x80x9cthe main disadvantage in the use of adenovirus as a gene transfer vector is that the viral vector generally remains episomal and does not replicate, thus, cell division leads to the eventual loss of the vector from the daughter cellsxe2x80x9d (Morgan, R. A., et al., Annual Review of Biochemistry 62:191-217 (1993)) (emphasis added).
Non-replication of the vector leads not only to eventual loss of the vector without expression in most or all of the target cells but also leads to insufficient expression in the cells that do take up the vector, because copies of the gene whose expression is desired are insufficient for maximum effect. The insufficiency of gene expression is a general limitation of all non-replicating delivery vectors. Thus, it is desirable to introduce a vector that can provide multiple copies of a gene and hence greater amounts of the product of that gene. The present invention overcomes the disadvantages discussed above by providing a tissue-specific, and especially a tumor-specific replicating vector, multiple DNA copies, and thus increased amounts of gene product.
Adenoviral vectors for recombinant gene expression have been produced in the human embryonic kidney cell line 293 (Graham, F. L. et al., J. Gen. Virol. 36:59-72 (1977)). This cell line, initially transformed with human adenovirus 5, now contains the left end of the adenovirus 5 genome and expresses E1. Therefore, these cells are permissive for growth of adenovirus 2 and adenovirus 5 mutants defective in E1 functions. They have been extensively used for the isolation and propagation of E1 mutants. Therefore, 293 cells have been used for helper-independent cloning and expression of adenovirus vectors in mammalian cells. E1 genes integrated in cellular DNA of 293 cells are expressed at levels which permit deletion of these genes from the viral vector genome. The deletion provides a nonessential region into which DNA may be inserted. For a review, see, Young, C. S. H., et al. in The Adenoviruses, Ginsberg, H. S., ed., Plenum Press, New York and London (1984), pp. 125-172.
However, 293 cells are subject to severe limitations as producer cells for adenovirus vectors. Growth rates are low. Titres are limited, especially when the vector produces a heterologous gene product that proves toxic for the cells. Recombination with the viral E1 sequence in the genome can lead to the contamination of the recombinant defective virus with unsafe wild-type virus. The quality of certain viral preparations is poor with regard to the ratio of virus particle to plaque forming unit. Further, the cell line does not support growth of more highly deleted mutants because the expression of E1 in combination with other viral genes in the cellular genome (required to complement the further deletion), such as E4, is toxic to the cells. Therefore, the amount of heterologous DNA that can be inserted into the viral genome is limited in these cells. It is desirable, therefore, to produce adenovirus vectors for gene therapy in a cell that cannot produce wild-type recombinants and can produce high titres of high-quality virus.
It is also desirable to control the replication of a therapeutic vector by other than endogenous cellular factors present within the cells to be treated. A high degree of replication could be disadvantageous to the patient, in that vector particles could be released into the bloodstream. This could have toxic side effects in tissues clearing the virus, such as kidneys, liver, lung and spleen, even if the vector does not replicate in non-treated tissue. Furthermore, while current therapeutic vectors have not been shown to replicate at low exposure to normal cells, a high exposure, as potentially caused from a high degree of replication and release of virus at the site of the treated tissue, may cause replication even in normal cells. For example, adenoviruses devoid of E1a, given in a sufficiently high dose, will replicate in normal cells.
Accordingly, it would be highly advantageous to be able to control the level of replication by adding a compound that could dampen replication if replication were excessive.
Another problem in the art is providing more than one gene product controlled by a heterologous regulatory sequence, for example a promoter, on a viral vector (such as cytokines, TK, or other cytotoxic genes, or heat-shock proteins). Thus, in a specific milieu, a particular combination of genes may offer therapeutic advantage. Accordingly, it would be desirable to have several genes in one vector and specifically expressed in the cells to be treated, for example, in a tumor cell. A limitation, however, is that one cannot provide multiple copies of the same promoter to drive each gene, because space would be wasted and the excess nucleotide stretches might not even be accommodated by the virus. Furthermore, during the cloning and replication, homologous recombination could produce deletions between identical promoters and therefore destroy the vector.
In view of the limitations discussed above, a general object of the invention is to provide novel expression vectors for tissue-specific vector replication and gene expression from the replicating vector.
Accordingly, the invention is directed to an expression vector that contains at least one gene that is essential for replication, which gene is operably linked to a heterologous transcriptional regulatory sequence (heterologous with respect to the gene essential for replication and/or to the vector type), such that an expression vector is created whose replication is conditioned upon the presence of a trans-acting transcriptional regulatory factor(s) that permits transcription from the transcriptional regulatory sequence, or the absence of a transcriptional regulatory factor(s) that normally prevents transcription from that transcriptional regulatory sequence. Thus, these regulatory sequences are specifically activated or derepressed in the target cell or tissue so that replication of the vector proceeds in that cell or tissue and expression of heterologous genes is induced or amplified.
Another object of the invention is to provide an expression vector whose replication, and hence gene expression, can be modulated.
Accordingly, a vector is provided that contains a gene encoding a gene product that can affect the rate and extent of vector replication.
Another object of the invention is to provide a way to coordinate and amplify the expression of multiple genes on a single vector.
Accordingly, an expression vector is provided in which the expression of multiple genes on the vector can be controlled and coordinated through the expression of a gene that is essential for replication, so that the expression of each of the multiple genes is conditional upon vector replication, replication depending upon the tissue-specific expression of the gene product.
Another object of the invention is to provide tissue-specific treatment of abnormal tissue. Thus, a further object of the invention is to provide a method to selectively distribute a vector in vivo in a target tissue, such that a greater number of cells contain the vector than would with a non-replicating vector, and spread of the vector is avoided or significantly reduced in non-target tissue.
Accordingly, a method is provided for selectively distributing a vector in a target tissue, by introducing the replication-conditional vector of the present invention into a target tissue that allows modulatable replication of the vector.
For providing tissue-specific treatment, another object of the invention is to selectively distribute a polynucleotide in a target tissue in vivo.
Accordingly, the invention is directed to a method for selectively distributing a polynucleotide in a target tissue in vivo by introducing the replication-conditional vectors of the present invention, containing the polynucleotide, into the target tissue that allows modulatable replication of the vector. The polynucleotide includes the entire vector or parts thereof, such as one or more heterologous genes.
For providing tissue-specific treatment, a further object of the invention is to selectively distribute one or more heterologous gene products in a target tissue.
Accordingly, the replication-conditional vectors of the present invention are constructed so that they contain one or more heterologous DNA sequences encoding a gene product that is expressed from the vector. When the vectors replicate in the target tissue, effective quantities of the desired gene product are also produced in the target tissue.
Another object of the invention, especially where tissue-specific treatment is involved, is to be able to modulate vector replication, that is, control the level of vector replication. When vector replication proceeds at levels that are undesirable, and particularly levels that may interfere with treatment, an object of the invention is to dampen or decrease the levels of replication by a desired degree. If the levels fall below the desirable amount, an object of the invention is also to be able to allow replication to increase to desirable levels by desired degrees.
Accordingly, the invention provides a method to modulate vector replication during treatment or otherwise (e.g., in producer cells), by providing a gene encoding a gene product that has the ability to interfere with vector replication. When replication is undesirably high, it can be decreased by means of this gene product.
Thus, the invention is further directed to vectors further containing a gene encoding a gene product that can be used to modulate vector replication.
Modulating replication also modulates the expression of heterologous genes contained in the vector. Thus, the expression of such genes can be induced, decreased, increased, or eliminated using the methods and vectors described herein.
Another object of the invention is to provide a method to identify abnormal tissue that can then be treated by the vectors of the present invention. Therefore, a further object of the invention is to identify a tissue in which the replication-conditional vectors of the present invention can be replicated by means of the heterologous transcriptional regulatory sequence contained on the vector so that the tissue can subsequently be treated with the vector, and in which tissue the replication can be modulated.
Accordingly, the invention is further directed to a method wherein the replication-conditional vectors of the present invention are exposed to a given abnormal tissue. If that tissue allows replication and modulation, then replication of the vector will occur and can be detected. Following identification of such a tissue, targeted treatment of that tissue can be effected by tissue-specific transcription and the consequent vector replication in that tissue in vivo.
Thus, a method is provided for assaying vector utility for tissue treatment comprising the steps of removing a tissue biopsy from a patient, explanting the biopsy into tissue culture, introducing a replication-conditional vector into the cells of the biopsy, and assaying for modulatable vector replication in the cells.
Another object of the invention is to provide producer cell lines for vector production. Preferably, the cell lines have one or more of the following characteristics: high titer virus production, resistance to toxic effects due to heterologous gene products expressed in the vector, lack of production of wild-type virus contaminating the virus preparation and resulting from recombination between integrated viral sequences and vector sequences, growth to high density and in suspension, unlimited passage potential, high growth rate, and by permitting the growth of highly deleted viruses that are impaired for viral functions and able to accommodate large pieces of heterologous DNA.
Accordingly, in a further embodiment of the invention, cell lines are provided containing the replication-conditional vector of the present invention, the cells of which allow modulatable replication of the vector or is deficient in a transcription-inhibiting factor(s) that prevents replication of the vector.
In further embodiments of the invention, the cell lines contain nucleic acid copies of the replicated vector. In other embodiments, the cell lines contain virions produced in the cell by replication in the cell of the replication-conditional vector.
In further embodiments, a method is provided for producing a replication-conditional vector or virion comprising the steps of culturing a producer cell line described above and recovering the vector or virion from the cells.
In still further embodiments, a method is provided for producing replication-conditional virions free of wild-type virions or viral vectors free of wild-type vectors, comprising the steps of culturing a producer cell line described above and recovering the replication-deficient virions or vectors from the cells.
In a preferred methods of treatment and diagnosis, the tissue is abnormally proliferating, and especially is tumor tissue. However, the methods are also directed to other abnormal tissue as described herein.
In a preferred embodiment of the invention, the replication-conditional vector is a DNA tumor viral vector.
In a further preferred embodiment, the DNA tumor viral vector is a vector selected from the group consisting of herpesvirus, papovavirus, papillomavirus, parvovirus and hepatitis virus vectors.
In a most preferred embodiment, the vector is an adenovirus vector.
However, it is to be understood that potentially any vector source is useful if it contains a gene essential for replication that can be operably linked to a tissue-specific transcriptional regulatory sequence.
In further methods of treatment and diagnosis, the vector is introduced into the cell or tissue by infection.
Replication can be vector nucleic acid replication alone or can also include virus replication (i.e., virion production). Thus, either DNA or virions or both may be distributed in the target tissue.
In a further preferred embodiment of the invention, a gene in the adenovirus E1 region is operably linked to the tissue-specific heterologous transcriptional regulatory sequence. Preferably, the E1a, E1b or E2a gene is operably linked to the tissue-specific transcriptional regulatory sequence.
In a further embodiment of the invention, the vector encodes one or more heterologous gene products. These heterologous gene products are expressed from the vector replicating in the target tissue. The heterologous gene product may be operably linked to its own promoter or may be operably linked to a transcriptional regulatory sequence from another gene. Regardless, the expression of the heterologous gene product may be controlled by E1a or another transactivator which in turn is regulated by the desired tumor-specific promoter.
In preferred embodiments of the invention, expression of one or more heterologous gene products depends upon expression of the gene essential for replication, which in turn is operably linked to the tissue-specific transcriptional regulatory sequence. Accordingly, when the gene essential for replication is expressed, it causes expression of one or more heterologous gene products, for example by transactivation. In this configuration, such a heterologous gene is activated/induced only when the vector replicates. This is because activation of the tissue-specific regulatory sequence causes expression of the gene product essential for replication which then causes both replication and activation of the expression of the one or more heterologous genes by its transactivation function. Accordingly, not only is expression of the heterologous gene activated, but expression is also amplified because when the vector replicates, each additional copy of the vector also contains a copy of the heterologous gene.
In a highly preferred embodiment, the E1a gene, being operably linked to a heterologous tissue-specific transcriptional regulatory sequence, controls the expression of one or more heterologous genes under the control of promoters that are transactivated by the E1a gene product. Thus, when the E1a gene is expressed, viral replication occurs and gene expression of one or more heterologous genes also occurs. Thus, expression of those genes is controlled at the level of replication and transactivation, such that an expression-amplifying effect is obtained.
In a further embodiment, one or more of the heterologous genes is operably linked to a tissue-specific regulatory sequence, such as the same transcriptional regulatory sequence to which the gene essential for replication is operably linked. Accordingly, the one or more heterologous genes are then activated only in a specific tissue. When the tissue-specific transcriptional regulatory sequence is the same one as that to which the gene essential for replication is operably linked, activation of the one or more heterologous genes occurs only when the vector replicates. Accordingly, as above, the one or more heterologous genes is both activated and amplified.
In other embodiments, the one or more heterologous genes is amplified when the vector replicates but not necessarily activated. This is when these genes are under the control of their own or other constitutive promoters. In this case, there is always a basal level of expression, but when the vector replicates, this expression is amplified because of expression from each new copy of the vector.
In the context of coordinate control (one or more heterologous genes under the control of the same transcriptional regulatory sequence, the same scenario of activation and amplification as discussed above applies.
In a further embodiment, the vector is not used to selectively destroy a cell or tissue type, such as a tumor, but is used to provide a gene product or products at specific levels that are physiologically desirable. An example of such gene product is insulin. Thus, using replication to control gene expression in order to achieve a very specific amount of gene expression is provided by the invention. Accordingly, the vector encodes a gene product which regulates the degree of virus replication, and hence gene expression of other gene products whose amounts must be controlled. An example of such product is one that would inhibit DNA polymerase or nucleotide synthesis. Accordingly, an excess of this product will down-regulate the vector replication. Gene products are also encompassed that will increase the level of vector replication if sufficient gene product is not produced.
Thus, according to the invention, one or more of the heterologous gene products is useful for modulating viral replication. It may directly or indirectly control viral replication as, for example, in the case of thymidine kinase in which viral replication is negatively affected and modulated by the addition of ganciclovir.
With respect to modulating gene expression, the vector provides for modulating the expression of heterologous genes in any of the configurations described above by modulating replication of the vector. This is preferably done via thymidine kinase or a gene product functioning as thymidine kinase does.
In a highly preferred embodiment, the thymidine kinase coding region is operably linked to the E3 promoter in an adenoviral vector. The E1a coding region is under the control of a heterologous tissue-specific promoter. Accordingly, when the tissue-specific promoter is activated, the E1a gene product is produced. This gene product then transactivates the E3 promoter, so that the TK gene is expressed. Further, since the E1a gene activates viral replication, and the E3 gene is not essential for replication, more copies of the viral vector are present to allow greater expression of the TK gene. Ganciclovir can then be added to modulate vector replication. If after adding the ganciclovir, replication falls below a desirable level, the amount of ganciclovir can be decreased to allow an increase in vector replication and so forth, so that desirable levels are permitted within any particular time frame. One example of desirable modulation is to decrease vector replication in a non-target cell.
In a further embodiment of the invention, a heterologous gene product is toxic for the target tissue.
In a further embodiment of the invention, the toxic heterologous gene product acts on a non-toxic prodrug, converting the non-toxic prodrug into a form that is toxic for the target tissue. Preferably, the toxin has anti-tumor activity or eliminates cell proliferation.
In preferred embodiments of the invention, the transcriptional regulatory sequence is a promoter. Preferred promoters include, but are not limited to, CEA, DF3, xcex1-fetoprotein, Erb-B2, surfactant, and especially lung surfactant, tyrosinase promoter, and endothelial-specific promoters. However, any genetic control region that controls transcription of the essential gene can be used to activate (or derepress) the gene. Thus, other genetic control elements, such as enhancers, repressible sequences, and silencers, can be used to regulate replication of the vector in the target cell. The only requirement is that the genetic element be activated, derepressed, enhanced, or otherwise genetically regulated by factors in the host cell and, with respect to methods of treatment, not in the non-target cell.
Preferred enhancers include the DF3 breast cancer-specific enhancer and enhancers from viruses and the steroid receptor family. Other preferred transcriptional-regulatory sequences include NF1, SP1, AP1, and FOS/JUN.
In further embodiments, promoters are not necessarily activated by factors in the target tissue, but are derepressed by factors present in the target tissue. Thus, in the target tissue, repression is lifted.
Transcriptional regulatory factors include, but are not limited to, transactivating factors produced by endogenous viral sequences such as from CMV, HIV, EBV, HSV, SV40, and other such viruses that are pathogenic in mammals and, particularly, in humans.
Methods for making such vectors are well known to the person of ordinary skill in the art. The art adequately teaches the construction of recombinant vectors with deletions or modifications in specific coding sequences and operable linkage to a heterologous transcription control sequence such that expression of a desired coding region is under control of the heterologous transcriptional regulatory sequence. Many viral sequences have been adequately mapped such that it is routine to identify a gene of choice and use appropriate well known techniques (such as homologous recombination of the virus with deleted or otherwise modified plasmids, or ligation of the two) to construct the vectors for tissue-specific replication and expression.